Intro to sample collection, slide preparation and interpretation of cytology samples from tissue masses (Proceedings)

Aspiration of a Mass

1. Hold the mass firmly with one hand.

2. Insert the needle, with attached syringe, into the mass.

(** Note: For small masses a more precise collection can be

accomplished by using a needle without an attached syringe**)

3. Pull back the plunger and hold to apply and maintain slight negative pressure.

4. The needle is then redirected in the mass several times while maintaining a negative pressure in the syringe.

5. Once material appears in the hub of the needle, the plunger is released and the needle can then be removed from the mass.

6. Disconnect the needle from the syringe and pull the plunger back.

7. Re-attach the needle to the syringe and depress the plunger to expel the aspirated material onto a clean glass slide.

8. A second glass slide is gently placed over the aspirated material and the material is allowed to diffuse out to form a thin layer between the two slides.

9. The two slides are gently slid apart forming a monolayer of aspirated cells.

10. The material is then allowed to air-dry prior to staining.

Slide Preparation

In cytology, cells that are properly smeared and stained can be described as "fried eggs" because of the similarity in the appearance of the nucleus and cytoplasm to the egg yolk and white. If the preparation is too thick, or is improperly stained, the cell outline may be seen, but intracellular detail will not be visible. These are the undesirable "hard-boiled eggs". Slide preparations that are smeared too thin will cause cell lysis and disruption of cellular architecture. These "scrambled eggs" are also undesirable. A properly prepared smear will have an area of "fried eggs" for viewing.

There are many stains available for routine cytologic evaluation in private practice. Most of these are Romanowsky-type stains such as Diff Quik™ . Alternatively, a true "Wright-Giemsa Stain Kit" from Volu-Sol, Inc. item number VWG-300 (www.volusol.com) is available for use in a clinical practice laboratory. This stain kit has the advantage of having a more consistent and better color distinction and better ability to stain mast cell granules. The exact staining procedure may vary depending on the stain used and manufactures recommendations should be used as general guidelines. However, for most Diff-Quik-type stains it is advisable to allow the slides to sit in the fixative for a minimum of 2 to 3 minutes before proceeding to the eosinophilic and basophilic stains. The number of dips in each stain will vary depending on the age of the stain and the thickness of the preparation, but usually 6 to 8 one-second dips in the eosinophilic stain and 5 to 6 one-second dips in the basophilic stain is sufficient. If the slide isunder-stained, it should be adjusted by returning the slide to the appropriate stain color. If the slide preparation is over-stained, the slide may be placed in methanol for several minutes to destain and then restained as before. The assessment of staining intensity is made by visualizing good color contrast between the cell nucleus and cytoplasm. The most important aspect of staining and slide preparation is to be able to visualize good nuclear and cytoplasmic detail. Without good slide preparation and staining, a reliable cytologic interpretation cannot be made.

Interpretation

The first step in making a cytological diagnosis is to classify the lesion into one of five general categories of disease processes: 1) inflammation, 2) cyst formation, 3) hemorrhagic lesion, 4) neoplasia, or 5) mixed cell population. In some cases more than one pathologic process may be occurring simultaneously in a single lesion. For instance, there may be hemorrhage within a neoplasm or inflammation within a cyst. However, when the population is not mixed and they are in their purist form, the categories are easily distinguished by the cell population present. The general cellular characteristics of the 5 categories of lesions are listed below.

1.Inflammation- characterized by the presence of neutrophils above what would be expected from any blood contamination

2. Cyst formation- large numbers of mature, keratinized, squamous epithelial cells oramorphous material of low cellularity.

3. Hemorrhagic lesion- blood in the presence of macrophages, some of which contain engulfed erythrocytes (erythrophagia) or hemosiderin.

4. Neoplasia- a homogeneous population of cells all from the same tissue of origin.

5. Mixed cell population- preparation contains both inflammatory (neutrophils, macrophages, etc.) and noninflammatory cells (epithelial or mesenchymal)

Inflammation

There are 3 types of inflammation that commonly occur, 1) Purulent inflammation,2) Pyogranulomatous inflammation,and 3) Eosinophilic inflammation. Each type will contain neutrophils as a major or minor component, however, they are distinguished from each other by the presence or absence of other cell types. The type of inflammatory response present may give some indication of the disease process that caused it.

Cyst Formation

There are 3 types of cutaneous cysts commonly seen in veterinary medicine, the epidermal inclusion cyst (follicular cyst), the apocrine cyst, and the sebaceous cyst. The cytologic and gross appearance of material aspirated from these lesions is described below. Other less common cysts may occur associated with various glandular structures of the body such as the mammary gland, prostate gland, or ovary. Cystic fluid drawn from glandular organs is typically of low cellularity, containing only low numbers of glandular epithelium in a proteinaceous background.

Hemorrhagic Lesion

Blood can be found in aspirates taken from almost any type of lesion. However, cytologically a diagnosis of hemorrhage is made by identifying hemosiderin pigment within macrophages or if whole erythrocytes are identified as being phagocytized by macrophages. Hemorrhage may be seen as a primary lesion, for example, in a hematoma induced by trauma or a bleeding disorder. Post-operative seroma formation will also appear as a proteinaceous fluid with evidence of hemorrhage. However, hemorrhage may alsobe a secondary component of a neoplastic process. Hemorrhage may be seen in a number of neoplasms, however, aspirates from hemangiomas and hemangiosarcomas, due to the low cellularity of the sample, may onlyyield evidence of blood and hemorrhage. The lesion is classified as hemorrhagic if hemorrhage is the only component of the lesion observed cytologically.

Neoplasia

Cytologically, neoplasia is characterized by the presence of a homogeneous population of cells that appear to have come from the same tissue of origin. This is best appreciated by the presence of cells with the same cytoplasmic characteristics. If a neoplasm is diagnosed, two important questions should be addressed; 1) is the lesion benign or malignant and 2) what is the tissue of origin of the neoplastic cell population.

Benign vs. Malignant

Primarily nuclear characteristics are used to make a determination of whether or not a neoplastic cell population is benign or malignant. Benign neoplasia or hyperplasia is characterized by the presence of a uniform population of cells. There should be uniformity in the cytoplasmic and nuclear size, shape, and nuclear to cytoplasmic ratio (N:C). Nucleoli may be seen in benign cells, however, if they are present they should be of consistent size, shape, and number between individual cells.

Malignant populations of cells display abnormal nuclear features that represent the cytologic criteria for malignancy. Commonly observed nuclear features of malignancy include: 1) anisokaryosis(a variation in nuclear size of 1.5 times or greater), 2) pleomorphism(variability in shape between nuclei in a cell population), 3) high or variable N:C ratio(normal, non-lymphoid cells have a N:C ratio of 1:3 to 1:8; ratios of 1:2 or higher suggest malignancy), 4) increased mitotic activity (mitotic figures are uncommon in normal tissues), 5) nucleoli that vary in size, shape, and numberbetween cells (variation within the same nucleus is especially significant, as is angular or pointed nucleoli), 6) coarse chromatin(nuclear material is no longer diffusely scattered throughout the nucleus, but gives the appearance of agglutination, 7) nuclear molding(compression and deformation of nuclei by other nuclei within the same or adjacent cells), and 8) multinucleation(the presence of 3 or more nuclei within one cell). Multinucleation is a normal feature of macrophages and is often seen in chronic pyogranulomatous inflammation. However, it may also be seen in populations of malignant cells particularly, but not exclusively, histiocytic tumors, bone tumors, and plasmacytomas

Categories of Neoplasia

In addition to determining if a neoplasm is benign or malignant, it is useful to classify tumors according to the tissue of origin. There are four categories of neoplasms identifiable by differences in cytologic appearance; epithelial, mesenchymal, round cell, and neuroendocrine.

Epithelial Neoplasms

Epithelial neoplasms exfoliate in clumps or sheets. They are very adherent to each other with extensive contact between adjacent cells and clear, linear zones between cells at areas of cell junctions. The cytoplasmic borders are very distinct and the nuclei are round to polygonal. Intracytoplasmic, secretory vacuoles and/or acinar formation may be seen in cell preparations taken from tumors originating from glandular epithelium. Malignancies of epithelial origin are termed carcinoma (nonglandular origin) or adenocarcinoma (glandular).

Mesenchymal Neoplasms

Aspirates taken from mesenchymal neoplasms are often less cellular than those taken from epithelial tumors. The cells are usually arranged individually, however, small clusters of mesenchymal cells can be held together by a stringy, eosinophilic material (collagen). The cytoplasm of mesenchymal cells is wispy and spindle shaped with tags of cytoplasm trailing off in 1 or 2 directions from the nucleus. The cell borders are often indistinct and nuclei are usually oval to polygonal in shape. Malignancies of mesenchymal origin are termed sarcoma.

Round Cell Neoplasms

Round cell neoplasms usually exfoliate well, and cytology is very useful in the definitive identification of these tumors. As a group, round cell tumors exfoliate as individually arranged cells with discrete round to oval cytoplasmic borders. There are 6 types of round cell neoplasms: histiocytic tumors, lymphoma, mast cell tumor, transmissible venereal tumor, plasma cell tumor, and melanoma. Each are very distinctive neoplasms, and they are easily identified cytologically. However, the cytologic appearance of these tumors, with regard to the nuclear criteria of malignancy, cannot be used as the sole means of predicting the biological behavior.

Neuroendocrine /Endorcine Neoplasms

Neuroendocrine /endocrine tumors are tumors of chemoreceptors (carotid and aortic bodies) or endocrine glands such as the thyroid, parathyroid, pancreas, and adrenal gland. These tumors share a characteristic cytologic feature. Slide preparations appear as free or "naked" nuclei embedded in a background of cytoplasm. Few distinct cytoplasmic borders are visualized. In general, neoplasms from this category may not have significant criteria of malignancy, and caution must be used when interpreting malignant potential of these lesions based on appearance alone. The thyroid tumors are the neuroendocrine tumors most frequently evaluated.

Canine thyroid tumors are usually located on the neck or near the thoracic inlet. Aspirates from thyroid tumors contain clumps of epithelial cells that appear as free nuclei embedded in a background of pale blue cytoplasm with infrequent visualization of cytoplasmic membranes or borders. Amorphous pink material (colloid) may be associated with some cell clusters. Dark, blue-black pigment (tyrosine granules) is sometimes seen in the cytoplasm of epithelial cells. Most thyroid tumors, even adenocarcinomas, will be composed of a fairly uniform population of cells, having few if any criteria of malignancy. However, in the dog over 90% of the clinically apparent thyroid tumors are adenocarcinomas. In contrast, most thyroid tumors in the cat are benign adenomas or adenomatous hyperplasia. Adenocarcinomas may occur in the cat, but are uncommon.

Inflammation and Neoplasia(The Mixed Cell Population)

The malignant criteria must be interpreted with caution when inflammation is present. Inflammation causes reactive changes in epithelial and mesenchymal cells that mimic malignancy. Unless ulcerated or necrotic, most mesenchymal tumors are not infiltrated with inflammatory cells. Therefore, in many cases, when a mixed population of anaplastic mesenchymal cells is seen along with neutrophils and/or macrophages, a reactive fibroplasia should be suspected until confirmed otherwise by histologic evaluation. Tumors associated with inflammation may include squamous cell carcinomas, nasal tumors, and bladder tumors. Most lesions with mixed cell populations will require histopathology to confirm a diagnosis.